The long-term objective of this project is to develop a novel dual-targeting therapeutic strategy (MDM2 and NFAT1) for the treatment of advanced breast cancer. Patients with advanced breast cancer, especially those with triple negative breast cancer (TNBC), cannot benefit from the recently-developed targeted therapies. Accumulating evidence has demonstrated that the dysfunction of several key signaling pathways, including NFAT1-MDM2 pathway, is associated with a poor prognosis and chemo-/radio-resistance, providing novel molecular targets for breast cancer therapy. The MDM2 oncogene is a major negative regulator of p53 and also has p53-independent oncogenic activity. It has been suggested to be a valid molecular target for breast cancer therapy. To date, the majority of small molecule inhibitors (SMIs) of MDM2 have been designed to block the binding of MDM2 to p53, and their anticancer activity was therefore dependent on the expression of wild-type p53 in the cancer cells. However, the majority of TNBC harbors mutant p53 and has high levels of MDM2, so the existing MDM2 SMIs are expected to have low or no efficacy against this subtype of breast cancer. Therefore, it is highly desirable to discover and develop novel MDM2 inhibitors that have direct effects on MDM2 and exert their anticancer activity independent of the p53 status of the cancer cells. The activation of NFAT1 is also commonly observed in breast cancers, especially in TNBC, promoting the development of an invasive, high-grade, and late-stage tumor phenotype. Therefore, the applicants propose NFAT1 as a novel target for the development of anti-breast cancer agents. Thus far, there has been no specific NFAT1 inhibitors developed for cancer therapy. Specific to this proposal, the applicants have recently discovered that NFAT1 up-regulates MDM2 expression and contributes to MDM2 overexpression in cancer cells and cancer tissues, including breast cancer. Based on newly-generated preliminary data, the applicants propose simultaneously targeting NFAT1 and MDM2 as a promising therapeutic strategy for advanced breast cancer. The applicants have recently accomplished a high-throughput screening of a natural product-based library, and have identified a series of disesquiterpenoid candidates with potent inhibitory effects on both NFAT1 and MDM2. One of the lead compounds, Japonicone A (JapA), has shown significant in vitro activity, in vivo efficacy, and minimal host toxicity in breast cancer models. Mechanistically, JapA directly binds to the MDM2 and NFAT1 proteins with high affinity, and induces MDM2 and NFAT1 protein instability. It also inhibits NFAT1-mediated MDM2 transcription by disrupting the binding of NFAT1 to the MDM2 P2 promoter. This first-in-class MDM2 inhibitor is distinct from all of the existing MDM2 inhibitors. In this revised proposal, the applicants will use JapA as a lead compound to test the central hypothesis that the dual targeting MDM2 and NFAT1 represents an effective and safe strategy for the treatment for advanced breast cancer. Three hypothesis-driven specific aims are proposed: 1) To demonstrate the therapeutic potential of JapA for advanced breast cancer; 2) To elucidate the molecular mechanisms by which JapA elicits anti-breast cancer activity as a specific NFAT1-MDM2 dual inhibitor; and 3) To characterize the pharmacological and toxicological properties of JapA. Upon completion of the proposed studies, the anticipated results will provide critical information about the therapeutic efficacy and safety of JapA and the value of simultaneously targeting MDM2 and NFAT1 in breast cancer. This project is highly significant, has high translational potential, and will generate a novel clinical candidate for breast cancer therapy, which would have a major impact on patient care and public health.